Platelet activating factor antagonists, named &#34;the phomactins&#34;, their preparation and use

ABSTRACT

A series of new compounds, called &#34;the phomactins&#34; is provided and may be isolated from the culture broth of fungi of the genus Phoma. especially Phomas sp. SANK 11486 (No. FERM BP-2598). These compounds are platelet activating factor antagonists and may be used for the treatment of disorders for which known such agents may also be used.

This application is a division of application Ser. No. 07/596,580 filedOct. 10, 1990 now U.S. Pat. No. 5,151,529, issued Sep. 29, 1992, whichis a continuation-in-part of application Ser. No. 07/440,235 filed Nov.22, 1989, now abandoned.

BACKGROUND TO THE INVENTION

This application is a division of application Ser. No. 07/596,580 filedOct. 10, 1990, now U.S. Pat. No. 5,151,529, issued Sep. 29, 1992, whichis a continuation-in-part of application Ser. No. 07/440,235 filed Nov.22, 1989, now abandoned.

The present invention relates to a series of new compounds, to which wehave assigned the class name "Phomactin". These compounds may beproduced by cultivation of an appropriate microorganism (fungus) of thegenus Phoma. The invention also provides a fermentation process forproducing these compounds using a novel strain of microorganism of thegenus Phoma, as well as the new strain itself, and provides methods andcompositions using the new compounds of the invention as plateletactivating factor antagonists.

"Platelet activating factor" is herein abbreviated, as is conventional,to "PAF".

Natural PAF, at least as isolated from mammalian tissues, is a mixtureof from 2 to 5 phospholipids, the number depending upon the nature ofthe original tissue. The major constituents of PAF may be represented bythe formula (A): ##STR1## in which R represents a log chain aliphatichydrocarbon group, which may be saturated or unsaturated. Natural PAF islevorotatory and the various components of natural PAF may beidentified, for example as: l-C_(16:0) =formula (A) where R represents ahexadecyl group: l-C_(18:0) =formula (A) where R represents an octadecylgroup: or l-C_(18:1) =formula (A) where R represents a 9(Z-octadecenylgroup. The convention used above for identifying the constituents of PAFgives the rotation first (l, in the above examples), followed by thenumber of carbon atoms, and finally the number of double bonds.

PAF exhibits a strong platelet activating and aggregating effect, fromwhich it derives its name. It has, however, in recent years been seen tobe a potentially crucial mediator in a wide variety of pathologicalprocesss. Thus, it also has a hypotensive effect and increasesvasopermeability; it is believed to be an active agent in the inductionof the shock state (for example endotoxin-induced shock) and to act as amediator of inflammatory disease. It has also been found to play a rolein cardiac and systemic anaphylaxis, gastric and intestinal ulceration,psoriasis and immune and renal disorders.

It is not, therefore, surprising that, as a result, PAF antagonists havebeen investigated with a view to developing new types of treatment forthe above pathologies, and notably new types of anti-shock agent andanti-inflammatory agent. Accordingly, various compounds have beeninvestigated in an attempt to find such PAG antagonists, and, currently,several compounds are known as PAF antagonists. Although the chemicalstructure of known PAF antagonists varies widely, and there appears tobe no obvious common factor linking their chemical structures, ingeneral, known materials having PAF-antagonist activity may beclassified according to their chemical structure as either PAF type ornon-PAG type compounds. Of the known compounds. PAF type antagonists aremostly prepared by chemical synthesis, whilst non-PAF type antagonistsare mostly recovered from the secondary metabolites of plants andmicroorganisms. Examples of compounds of the non-PAF type include, forexample, ginkgolide (isolated from Ginkgo biloba), kadzurenone (isolatedfrom Piper futokadzura), veraguensin (isolated from Magnolia acuminata),galbengin and galbrabin (isolated from Himantandra velgravena),nectandrin A and B (isolated from Nectandra rigida), burseran (isolatedfrom Bursera microphlla) [details of the above compounds are given by P.Braquet and J. J. Godfroid: Trends in Pharm. Sci., Vol. 7, pp. 397 etseq (1986)], a gliotoxin derivative [isolated from Penicilliumterlikowskii: M. Okamoto, Chem. Pharm. Bull., 34, 340 (1986)] and adiketopiperazine derivative [isolated from Streptomyces sp.: S. Takase,J. Org. Chem., 52, 3485 (1987)].

A variety of PAF-type compounds are known, but these are not relevant tothe present invention.

A review of the nature and uses of PAF antagonists is given by P.Braquet et al., Trends in Pharm. Sci., Vol. 10, pp. 23 et seq (1989).Braquet et al examined the effect of BN-52021 (5 mg kg⁻¹, given 15 minbefore coronary artery occlusion) on arrhythmias in mongrel dogs. Asshown in Table I, BN-52021 significantly reduced the number of ectopicbeats during the 30 min ischemic period. In addition, preliminarystudies in rats subjected to coronary artery ligation have shown thatBN-52021 (5 mg kg⁻¹, i.v. immediately before occlusion), significantlyreduced the infarct size as assessed by histological examination after24 h of occlusion. In the control group the infarct size was 26±2.2% ofheart tissue, whereas in the BN-52021-treated group this value wasreduced to 17±1.8% (p<0.05). ##STR2##

                                      TABLE I                                     __________________________________________________________________________    Effect of BN-52021 on arrhythmias evoked by ligation of left anterior         descending                                                                    coronary artery in dogs anesthetized with halothane                                   Number of ectopic beats during 30 min of ischemia, mean ± SEM      Treatment                                                                           η                                                                           5-10  10-15 15-20 20-25  25-30 min                                    __________________________________________________________________________    Control                                                                             18                                                                              106 ± 26.0                                                                       119 ± 33.4                                                                       208 ± 41.8                                                                       355 ± 69.5                                                                        473 ± 104.8                               BN-52021                                                                             7                                                                               10 ± 5.4*                                                                        10 ± 5.3*                                                                        15 ± 6.3*                                                                        55 ± 28.6*                                                                        98 ± 39.0*                               __________________________________________________________________________     These cumulative data represent the sum of single ventricular                 extrasystoles in salvos, and ventricular tachycardia. BN52021 was             introduced in an i.v. bolus of 5 mgkg.sup.-1 15 min before coronary arter     ligation.                                                                     *ρ < 0.05 (Wilcoxon test).                                           

We have now discovered a series of new PAF-antagonist compounds of thenon-PAF type, which we have in accordance with current convention, named"the phomactins", and which can be produced by the cultivation of amicroorganism, which we have identified as SANK 11486, which belongs tothe species Phoma and which has been isolated from the shells of certaincrabs.

BRIEF SUMMARY OF INVENTION

In accordance with the present invention, there are provided, as newcompositions of matter, novel compounds which we have called thephomactins.

The phomactins are here defined as those compounds having PAF antagonistactivity and capable of production by the fermentation of Phoma sp. SANK11486. In general, the chemical structure of the phomactins is believedto be characterised by a cyclohexene ring which is bridged between acarbon atom of the double bond and a carbon atom 2 positions removedtherefrom by a 9 membered carbon bridge structure, which itself may alsobe bridged.

The invention also provides a process for producing a phomactin by thecultivation of a phomactin-producing microorganism of the strain Phoma,and separating at least one phomactin from the cultured broth.

The invention also provides a pharmaceutical composition for thetreatment of inflammation or shock, comprising a PAF antagonist incombination with a pharmaceutically acceptable carrier or diluent,wherein the PAF antagonistis at least one phomactin, as defined above.

The invention will further provides a method of treating a PAF-mediatedpathology in a mammal susceptible to such pathology by administering tosaid mammal an effective amount of a PAF antagonist selected from thegroup consisting of the phomactins, especially phomactins A and B.

The invention still further provides a method for the treatment orprophylaxis of asthma, inflammation or shock comprising administering anamount of a PAF antagonist to an animal (which may be a mammal, e.g.human) sufficient to effect treatment or prophylaxis of inflammation orshock, wherein said PAF antagonistis at least one phomactin, as definedabove.

DETAILED DESCRIPTION OF INVENTION

One of the compounds of the present invention, which we have called"Phomactin A", has the following chemical formula and properties:

1) Chemical formula as shown in formula (I): ##STR3##

2) Appearance: Colorless oily substance.

3) Specific rotation: [α]_(D) ²⁵ =+146.7° (c 0.75, chloroform).

4) Elemental analysis:

    ______________________________________                                        Found:       C, 71.65%; H, 9.10%.                                             Calculated:  C, 71.82%; H, 9.04%.                                             ______________________________________                                    

5) Molecular formula: C₂₀ H₃₀ O₄ (Determined by high resolution massspectroscopy).

6) Molecular weight: 334 (Determined by mass spectroscopy).

7) Infrared absorption spectrum, ν_(max) cm⁻¹ (liquid film): 3395, 1580,1450, 1380, 1230, 1050, 960 and 756.

The infrared absorption spectrum measured as a liquid film is shown inFIG. 1 of the accompanying drawings.

8) ¹ H-Nuclear magnetic resonance spectrum, δ ppm:

The nuclear magnetic resonance spectrum (500 MHz) measured inperdeuterated methanol using tetramethylsilane as an internal standardis shown in FIG. 2 of the accompanying drawings.

9) ¹³ C-Nuclear magnetic resonance spectrum, δ ppm:

The nuclear magnetic resonance spectrum (126 MHz) measured inperdeuterated methanol using tetramethylsilane as an internal standardis shown in FIG. 3 of the accompanying drawings, and the chemical shiftsand multiplicities are shown below:

14.9 (q), 16.5 (q), 19.6 (q), 21.9 (q), 25.8 (t), 27.8 (d), 34.2 (t),34.5 (t), 37.6 (t), 38.0 (s), 38.6 (t), 62.6 (d), 72.0 (t), 74.6 (d),81.2 (s), 110.0 (s), 128.7 (s), 131.3 (s), 131.4 (d), 144.6 (s).

(q is quartet, t is triplet, d is doublet and s is singlet).

10) Ultraviolet absorption spectrum, λ_(max) nm:

The ultraviolet absorption spectrum measured in ethanol shows endabsorption only.

11) Solubility:

Soluble in methanol, ethanol, acetone, ethyl acetate, chloroform,dimethyl sulfoxide, benzene and diethyl ether; and insoluble in water.

12) Color reactions:

Color tests with sulfuric acid and iodine are positive.

13) Thin layer chromatography:

Rf: 0.54.

Adsorbent: Silica gel plate (Kieselgel 60F₂₅₄, Merck).

Developing solvent: a 1:2 by volume mixture of hexane and ethyl acetate.

The systematic name of phomactin A is3,3a-dihydroxy-2,6-(3'-methyl-3'-hexeno)-2,6,7-trimethyl-3a,5,6,7,8,8a-hexanhydrofuro[2,3,4-de]chroman.

Another of the compounds of the present invention, which we have called"Phomactin B", has the following chemical formula and properties:

1) Chemical formula as shown in formula (II): ##STR4##

14) Appearance: Colorless needles, melting at 180°-182° C.

3) Specific rotation: [α]_(D) ²⁵ =+175° (c 0.70, chloroform).

4) Elemental analysis:

    ______________________________________                                        Found:       C, 71.71%; H, 9.09%.                                             Calculated   C, 71.82%; H, 9.04%.                                             ______________________________________                                    

5) Molecular formula: C₂₀ H₃₀ O₄ (Determined by high resolution massspectroscopy).

6) Molecular weight: 334 (Determined by mass spectroscopy).

Infrared absorption spectrum, ν_(max) cm⁻¹ (KBr): 3420, 3380, 1669,1630, 1460, 1392, 1250, 1200, 1080, 1000, 903 and 812.

The infrared absorption spectrum measured on a KBr disk is shown in FIG.4 of the accompanying drawings.

¹ H-Nuclear magnetic resonance spectrum, δ ppm:

The nuclear magnetic resonance spectrum (500 MHz) measured inperdeuterated methanol using tetramethylsilane as an internal standardis shown in FIG. 5 of the accompanying drawings.

¹³ C-Nuclear magnetic resonance spectrum, δ ppm:

The nuclear magnetic resonance spectrum (126 MHz) measured inperdeuterated methanol using tetramethylsilane as an internal standardis shown in FIG. 6 of the accompanying drawings, and the chemical shiftsand multiplicities are shown below:

14.5 (q), 16.4 (q), 19.7 (2×q), 22.7 (t), 23.2 (q), 33.6 (t), 36.7 (t),37.4 (t), 41.5 (s), 46.3 (d), 62.8 (s), 65.9 (d), 71.4 (d), 73.3 (s),120.3 (d), 135.6 (d), 136.8 (s), 147.2 (s), 200.1 (s)

(abbreviations as above).

10) Ultraviolet absorption spectrum, λ_(max) nm:

The ultraviolet absorption spectrum measured in ethanol shows anabsorption at 240 nm (E=2,500).

11) Solubility:

Soluble in methanol, ethanol, acetone, ethyl acetate, chloroform,dimethyl sulfoxide, benzene and diethyl ether; and insoluble in water.

12) Color reactions:

Color tests with sulfuric acid and iodine are positive.

13) Thin layer chromatography:

Rf: 0.51.

Adsorbent: Silica gel plate (Kieselgel 60F₂₅₄, Merck).

Developing solvent: a 1:2 by volume mixture of hexane and ethyl acetate.

The systematic name of phomactin B is13,15-dihydroxy-3,4-epoxy-2-oxo-4,8,11,12,15-pentamethylbicycl[9.3.1]pentadeca-7,14-diene.

The phomactins of the present invention can exists in the form ofvarious stereoisomers and geometric isomers, although, in the form asnaturally produced by a microorganism of the genus Phoma, the compoundsof the present invention will normally exist as a single isomer. In theformulae given above, the individual isomers and mixtures of the isomersare represented by a single formula. However, the present inventioncovers not only the individual isomers but also mixtures of any two ormore isomers, whether naturally produced or produced by chemicalmanipulation. Where the naturally produced isomer is not the onedesired, the desired isomer may be produced by chemical means, as iswell known in the art.

It is currently believed that the configurations of phomactins A and Bare as shown in formulae (Ia) and (IIa), respectively: ##STR5##

The phomactins have been produced by the cultivation of a strain ofmicroorganism of the genus Phoma (a fungus), which we have designatedstrain SANK 11486. This phomactin-producing microorganism of the strainSANK 11486 employed in the present invention was isolated by us from theshell of a crab. Chinoecetes opilio (colloquially called "susugani",which is a crab whose shell turns black) collected in the vicinity ofthe coast at Fukui prefecture, Japan.

The microbiological properties of the strain are as follows:

Growth on oatmeal agar at 25° C. reached a 20 mm diameter in 7 days and38 mm in 14 days. On both of the these days, the colonies had a wetvelvety appearance and a greenish gray color.

On the oatmeal agar, only mycelia were observed, but no spores orconidia; however, on oatmeal agar prepared with an artificial sa water(Brand name: Jamarin S) it forms pycinidia in which conidia areproduced.

The pycnidium is observed as a semispherical or pyriform shape ofdimensions 80-120×80-150 μm and it has a hole in the center thereof. Thediameter of the hole is 5-15 μm and there are brown setae of dimension20-50×2-4 μm on the periphery.

The conidiophore is not particularly differentiated, and the inner mostlayer of phore wall became phialide. The condiophore is colorless,single cellular, elliptical or elongated elliptical of dimensions2.5-4.0×2.0-2.5 μm.

The strain does not grow at 37° C.

Based upon the above features, and referring to standard texts [B.C.Sutton, "The Coleomycetes", Commonwealth Mycological Institute, 19880;and J. Kohlmeyer and E. Kohlmeyer, "Marine Mycology, The Higher Fungi",Academic Press, 1979] the present strain was identified as a new strainof a known species, Phoma sp., and was given a stock number SANK 11486(No. FERM P-10364). The color expressions and codes used herein wereassigned according to the directions in the Methuen Handbook of Color,3rd Ed. (1978), written by A. Kornerup and J. H. Wanscher, and publishedby Eyre Methuen, London.

The strain was deposited by us at the Fermentation Research Institute,Agency of Industrial Science and Technology, Tokyo, Japan with theaccession No. FERM P-10364 and was redeposited under the conditionsprescribed by the Budapest Treaty on 20 September 1989 under theaccession No. FERM BP-2598.

It has been established that strain SANK 11486 produces the phomactins.However, as is well known, the properties of fungi in general can varyconsiderably and such fungi can readily undergo mutation, both throughnatural causes and as the result of induction by artificial means.Accordingly, the process of the present invention embraces the use ofany microorganism which can be classified within the genus Phoma,especially strains of species of the genus Phoma to which the new strainSANK 11486 belongs, and which shares with strain SANK 11486 thecharacteristic ability to produce the phomactins. It is a matter ofsimple experimentation to determine, on the basis of the informationgiven herein regarding the properties of the phomactins, notably thebiological properties, whether any given strain produces any of thephomactins or produces it or them in sufficient quantity to render thatstrain of potential commercial interest.

The novel compounds, the phomactins, of the present invention may beprepared by the cultivation of any of these strains of fungus in culturemedia of the type conventionally used for the production of otherfermentation products from similar microorganisms. Such medianecessarily contain microbiologically assimilable salts, as is wellknown to those skilled in the art. In all respects, however, the culturemedia employable in the present invention are the same as those knownfor the cultivation of known strains of fungi.

Preferred examples of carbon sources include: glucose, fructose,maltose, sucrose, mannitol, glycerol, dextrin, oats, rye, corn starch,potato, potato starch, corn meal, soybean meal, cottonseed cake,cottonseed oil, molasses, citric acid, tartaric acid and the like,preferably glucose, sucrose or potato or any combination thereof. Suchcompounds can be used alone or in any suitable combination. In generalthe amount used may vary in the range of from 1 to 10% by weight of theculture medium.

Preferred nitrogen sources are normally protein-containing materials,but they may be other materials, including inorganic materials, such asare commonly used in a fermentation process. Examples of such nitrogensources include: soybean meal, wheat bran, peanut meal, cottonseed cake,cottonseed oil, cottonseed meal, casein hydrolyzates, pharmamin, fishmeal, corn steep liquor, peptone, meat extract, yeast, yeast extract,salt extract, sodium nitrate, ammonium nitrate, ammonium sulfate and thelike, preferably peptone and/or meat extract. These nitrogen sources maybe used alone or in any suitable combination. In general, we prefer toemploy them at a concentration between 0.2 and 6% by weight of theculture medium.

The nutritive inorganic salts that should be added to the culture mediumare conventional salts that are capable of providing various ionsnecessary to the growth of microorganisms, such as sodium, ammonium,calcium, phosphate, sulfate, chloride and carbonate ions. In addition,the medium should preferably contain minor amounts of other essentialmetals, such as potassium, calcium, cobalt, manganese, iron andmagnesium.

The aqueous component of the culture medium may, as illustrated incertain of the examples hereafter, be provided by a form of sea water,whether natural or (as is preferred) artificial; however, equally goodresults are achievable using ordinary water, and the use of a form ofseat water is, therefore, unnecessary to the present invention.

When the process of the present invention is carried out by a liquidculture technique, an antifoaming agent, such as a silicone oil, a plantoil or a surface-active agent, is preferably used in the culture medium.

The pH of the culture medium for producing the phomactins by thecultivation of Phoma sp. SANK 11486 preferably varies within the rangeof from 5.0 to 9.0 and is more preferably from 6.5 to 8.5.

The cultivation may be carried out at any temperature within the rangeof from 15° to 30° C., although a temperature of from 20° to 28° C. ispreferred for good growth, and a temperature of from 22° to 26° C. ispreferred in order to optimize the production of the phomactins.

The phomactins are produced under aerobic culture conditions andconventional aerobic culture methods. such as solid culture, shakingculture and aeration-stirring (submerged) culture methods, may be used.In the case of small scale cultivation, shaking culture for a period offrom several days to two weeks at a suitable temperature, such as22°-26° C., is typical. In such a small scale culture method, theculture may be initiated with 1 or 2 proliferation steps, producing seedcultures in, for example, Erlenmeyer flasks, fitted with baffle plates,which serve as a liquid flow regulator. The medium for the seed culturesteps preferably contains both carbon and nitrogen sources, as indicatedabove for the cultivation steps. In the preferred sequence of operationsfor such small scale cultivation, the seed culture flasks are shaken ina constant temperature incubator at, for example, 22°-26° C. for 7 daysor until sufficient growth is achieved. The grown seed culture is thentransferred to a second seed medium or to the production medium. When anintermediate growth phase is used, essentially the same method is usedfor growth, and an aliquot of the resulting intermediate product isinoculated into the production medium. The inoculated flask may beincubated for several days whilst shaking, at a suitable temperature,e.g. 22°-26° C., and, after completion of the incubation, the contentsof the flask may be centrifuged or filtered.

In the case of large scale production, the use of an appropriatefermentor equipped with a stirrer and an aeration apparatus ispreferred. In this case, the nutritive medium can be prepared inside thefermentor. The medium is preferably first sterilized by elevating thetemperature to sufficiently high temperature, such as 125° C.; aftercooling, the sterilized medium may be inoculated with the previouslyprepared seed culture. The culture then proceeds under stirring andaeration at a suitable temperature, e.g. 22°-26° C. This method issuitable for obtaining the compound of the present invention in a largeamount.

The progress of the cultivation and the amount of the phomactinsproduced as the culture proceeds can be determined by various well knowntechniques, which are conventionally used in this art. For example, itmay be monitored by following the increase in anti-PAF activity of theculture broth. Generally, the total amount of phomactins produced isbelieved to reach a maximum between 10 and 14 days after initiation ofthe fermentation, but the exact time will vary depending upon thetemperature and other fermentation conditions; however, the exactoptimum time for any given set of conditions can easily be determined bymonitoring the progress of the production of the phomactins, assuggested above.

After completion of the cultivation, the phomactins present in theliquid fraction of the cultured mass may be separated by filtration,preferably using a filter aid such as diatomaceous earth, or bycentrifugation or by any other standard technique. The phomactinspresent in the filtrate or in the supernantant can be recovered byextraction from this filtrate and/or they may be purified utilizing thephysiochemical properties of the phomactins. For example, the phomactinspresent in the filtrate or the supernatant can be extracted at a neutralpH with one or more water immisible organic solvents (such as diethylether, ehtyl acetate, hexane, chloroform, ethylene chloride or methylenechloride), and the solvents may be used individually or as a mixture.

An alternative method is to use an adsorption agent, for exampleactivated charcoal, or an adsorption resin, such as an Amberlite (trademark) XAD-2 or XAD-4 resin (Rohm and Haas Co., Ltd.), or a Diaion (trademark) HP-10, HP-20, CHP-20P or HP-50 resin (mitsubishi KaseiCorporation). The fluid containing the phomactins is passed through alayer of one of these adsorbents in order to remove the adsorbedimpurities or the adsorbed phomactins are eluted with a suitable eluent,such as aqueous methanol, aqueous acetone or aqueous butanol. Almost allof the phomactins produced is believed to be released into the culturemedium, and very little is thought to be retained in the cells; however,if it is desired to recover any phomactins that may be retained in thecells, this may be achieved by conventional means.

The phomactins obtained as described above can be further purified byvarious conventional techniques, notably the various chromatographytechniques, such as adsorption column chromatography using a carriersuch as silica gel or a magnesia-silica gel, such as Florisil (trademark), partition column chromatography using Sephadex (trade mark) LH-20(Pharmacia Fine Chemicals), or high performance liquid chromatographyusing a column for a normal or reverse phase process.

At any suitable stage in the above procedure, the several phomactins maybe separated from each other by conventional methods, of which thechromatography techniques, notably column chromatography, are the mosteffective. It is, therefore, convenient to separate the phomactins inthe course of the purification procedure outlined above.

The phomactins of the present invention are new compounds which have notpreviously been described in the literature. They have been shown toexert a PAF antagonist activity in test animals and are thereforeexpected to demonstrate a similar activity in other animals (e.g.humans, dogs, cats and rabbits). They are thus expected to be useful asan antithrombotic drug. and are also expected to be useful for theprophylaxis and therapy of inflammation, shock, asthma, peptic ulcers,renal failure, hypertension, ischemia and similar disorders which may berelieved by the administration of PAF antagonists.

Although it has not been established with certainty and we do not wishto be limited by any theory, it is currently believed that thephomactins operate as PAF antagonists by binding to PAF receptors inplace of PAF itself. This mode of operation appears to be common toseveral types of PAF antagonists, despite widely differing chemicalstructures.

One or more of the phomactins of the present invention may be used aloneas a therapeutic or prophylactic drug, or it or they may be mixed withpharmaceutically acceptable carriers and/or excipients and/or diluentsappropriate to its or their intended route of administration, as is wellknown in the art. For example, for oral administration, the drug may beemployed in the form of powders, granules, tablets or capsules; forparenteral administration, the drug may be employed in the form ofinjections. The dosage of the compounds of the present invention willvary, depending upon the severity and nature of the disease or disorder,the age, body weight and condition of the patient and upon the route,frequency and period of administration. However, a suitable daily doseof the phomactin or phomactins of the present invention for an adulthuman by an enteric route would be in the range of from 1 mg to 1,000mg, which may be administered as a single dose or as divided doses e.g.from one to several times per day.

The preparation of the compounds of the present invention is furtherillustrated by the following non-limiting Examples.

EXAMPLE 1 a) Cultivation

One loopful of Phoma sp. SANK 11486 from a slant culture was inoculatedinto a 500 ml Erlenmeyer flask fitted with baffles (seed flask) andcontaining 100 ml of a seed culture medium having the compositiondescribed below.

    ______________________________________                                        Composition of medium                                                         ______________________________________                                        Sucrose                  20     g                                             Potato                   100    g                                             Peptone                  10     g                                             Monobasic potassium phosphate                                                                          5      g                                             Artificial sea water     1,000  ml                                            (Brand name: Jamarin S,                                                       Jamarin Laboratories Co. Ltd.)                                                pH 8.5                                                                        ______________________________________                                    

The whole of the resulting culture broth was then cultivated at 26° C.for 7 days on a 200 rpm rotatory shaker (7 cm radius gyration).

100 ml of a culture medium (having the same composition as that of theseed culture medium) were put into each of 120×500 ml Erlenmeyer flasks,and the medium in each flask was sterilized. 3 ml of the seed culturewas then inoculated into each flask. The material in each of the 120flasks was then subjected to shaking culture under the same conditionsas were used for the seed culture for 7 days.

b) Isolation

The whole of the cultured broth from all 120 flasks was combined andfiltered with suction, to give 13 liters of a filtrate. This was thenextracted twice, each time with an equal volume of ethyl acetate. Theethyl acetate extracts were combined and washed with 5 liters of water;they were then dried over anhydrous sodium sulfate, after which theywere evaporated to dryness under reduced pressure using a rotaryevaporator, to give 360 mg of oil substance. The whole of this oilysubstance was purified by adsorption column chromatography through 30times its own volume of silica gel (Art. 9385, 230-400 Tyler standardmesh, Merck), eluted with a 1:1 by volume mixture of hexane and ethylacetate. The fractions showing PAF antagonist activity were collected,and the solvent was removed by distillation under reduced pressure togive 130 mg of oily material. The whole of this oily material wasfurther purified by reverse phase column chromatography (Lobar RP-8column, size B, Merck). The fractions eluted with 80% by volume aqueousmethanol were collected and the solvent was distilled off. The resultingresidual oily material was purified by high performance liquidchromatography (Senshu pack ODS-2251-S, Senshu Kagaku Co., Ltd.) using40% by volume aqueous acetonitrile as the eluent. The collectedfractions were freed from the solvent by distillation under reducedpressure to afford 3 mg of the desired compound, phomactin A {Chemicalname:(3'E)-3,3a-dihydroxy-2,6-(3'-methyl-3'-hexeno)-2,6,7-trimethyl-3a,5,6,6,8,8a-hexahydrofuro[2,3,4-de]-chroman},as a colorless oil.

EXAMPLE 2

The whole of the culture broth obtained by culturing a seen culture in480 flasks under the same conditions as described in Example 1 wasfiltered, with the aid of suction, and the resulting 48 liters offiltrate were twice extracted, each time with an equal volume of ethylacetate. The combined ethyl acetate extracts were washed with 20 litersof water and dried over anhydrous sodium sulfate, after which they wereevaporated to dryness under reduced pressure using a rotary evaporator,to give an oil substance. This oily substance was subjected to silicagel column chromatography through about 30 times its own volume ofsilica gel (Art. 9385, 230-400 mesh Tyler, Merck), eluted with a 1:1 byvolume mixture of hexane and ethyl acetate and then with a 2:3 by volumemixture of hexane and ethyl acetate. The first fraction was concentratedby evaporation under reduced pressure and purified by reverse phasecolumn chromatography (Lobar RP-8 column, size B, Merck) and highperformance liquid chromatography (Senshu pack ODS-2251-S, Senshu KagakuCo., Ltd.) as described in Example 1, to give 9.4 mg of phomactin A as acolorless oil. The second fractions eluted with the 2:3 mixture wereconcentrated by evaporation under reduced pressure to give 78.2 mg ofoil substance, which was further purified by silica gel columnchromatography (Lobar Si60 column, size A, Merck) eluted with a 1:1 byvolume mixture of hexane and ethyl acetate. The resulting activefractions were then subjected to reverse phase column chromatography(Lobar RP-8 column, size B, Merck) eluted with 60% by volume aqueousmethanol to give 15.9 mg of phomactin B {Chemical name:(7E)-13,15-dihydroxy-3,4-epoxy-2-oxo-4,8,11,12,15-pentamethylbicyclo[9,3,1]pentadeca-7,14-diene}as colorless needles.

EXAMPLE 3

A seed culture was cultured in 55 flasks at 23° C. for 11 days in asimilar manner to that described in Example 1, except that tap water wasused instead of artificial sea water. The whole of the resulting culturebroth was filtered, with the aid of suction, and 5.4 liters of theresulting filtrate was extracted twice with the same volume of ethylacetate. The combined ethyl acetate extracts were washed with water anddried over anhydrous sodium sulfate, after which they were evaporated todryness under reduced pressure using a rotary evaporator, to give 636 mgof an oily substance. The whole of this oil substance was subjected tosilica gel column chromatography (Art. 9385, 230-400 mesh Tyler, Merck)eluted with a 1:4 by volume mixture of hexane and ethyl acetate. Theactive fractions were concentrated by evaporation under reducedpressure, to give 274 mg of oily substance. This was further purified byhigh performance liquid chromatography (Senshu pack ODS-2251-S, SenshuKagaku Co., Ltd.) eluted with 80% by volume aqueous methanol at a flowrate of 6 ml/min to give 14.0 mg of phomactin A with a retention time of22.5 minutes. The fraction preceding the phomactin A-containing fractionwas concentrated by evaporation under reduced pressure, and purified byhigh performance liquid chromatography (Senshu pack ODS-2251-S, SenshuKagaku Co., Ltd.) eluted with 70% by volume aqueous methanol at a flowrate of 6 ml/min to give 14.4 mg of phomactin B with a retention time of18 minutes.

EXPERIMENT 1

The following antiplatelet aggregation test was performed, using thephomactin A thus obtained.

Blood was drawn from a rabbit heart and immediately mixed with ninetimes its volume of a 3.8% w/v aqueous solution of sodium citrate. Aplatelet-rich plasma (PRP) was then obtained as a supernatant bycentrifugation of the blood mixture at 150×g for 15 minutes at roomtemperature. The precipitated fraction remaining from the blood mixturewas then centrifuged for a further 15 minutes at 10,000×g to obtain aplatelet-poor plasma (PPP) as a supernatant. Appropriate proportions ofthe PRP and the PPP were mixed to obtain a plasma having a plateletcount of 6×10⁵ /μl.

Platelet aggregation was determined by the method of Born et al.[Nature, 194, 927-929 (1962)] where an increase in light transmissionthrough the test sample is measured by means of an aggregometer.

3 μl of a dimethyl sulfoxide solution containing the compound to betested at an appropriate concentration was added to 250 μl of a salinesolution of synthetic C_(16:0) PAF (at a concentration sufficient togive a final concentration of 1×10⁻⁸ -3×10⁻⁸ M) was added andaggregation was observed for 5 minutes. The aggregation resulting fromthe addition of the PAF alone, without the prior addition of the testcompound, was taken as 100%.

Instead of the test solution, physiological saline was added and theaggregation at one minutes after the addition of C₁₆ -PAF was used asthe control. As a result, phomactin A was found to inhibit plateletaggregation by about 91.7% at a concentration of 10 g/ml, whichcorresponds to an IC₅₀ (i.e. the concentration necessary to inhibitaggregation by 50%) of 17 μM.

The same experiment was carried out with phomactin B. whose IC₅₀ wasalso found to be 17 μM, indicating a good and useful ability to inhibitplatelet aggregation.

We claim:
 1. The compound of formula (II): ##STR6##
 2. The compound offormula (IIa): ##STR7##
 3. A pharmaceutical composition 1 of 2comprising the compound of claim 2 or 4 in combination with apharmaceutically acceptable carrier or diluent.